Nanopore-matched protein shuttle for molecular characterization and methodology for data analysis thereof

ABSTRACT

Systems and methods are provided for characterizing shuttle capture events in a nanopore sensor. The method first collects time-dependent current blockage signatures for at least one bias voltage. The method then identifies each signature as corresponding to a permanent or transient event. The method then generates a protein dynamics landscape (PDL) for the transient event signatures. The PDL comprises a set of histograms of nanopore current data and characterizes current through the nanopore during shuttle capture events. The method can then comprise identifying an entrance level blockage value based on the permanent event signatures. Permanent event captures can be determined by time duration which is larger than a certain threshold time value. Applying a between the fluidic chambers above a threshold voltage level can be used to control that the vast majority of events are permanent.

CROSS REFERENCED TO RELATED APPLICATIONS

This application is a 371 National Phase Entry of International PatentApplication No. PCT/US2018/042480 filed Jul. 17, 2018, which claimspriority to and the benefit of U.S. Provisional Patent Application No.62/533,215, filed Jul. 17, 2017 and entitled “Nanopore-Matched ProteinShuttle for Molecular Characterization and Methodology for Data AnalysisThereof,” the contents of which are hereby incorporated by reference intheir entirety as if fully set forth herein.

FIELD

The present invention relates to systems and methods for characterizinga nanopore sensor and for analyzing and visualizing single moleculemeasurements obtained with the sensor.

BACKGROUND

Molecules can be trapped and electrically monitored via a nanopore. Inparticular, protein shuttles, typically including a protein molecule andother molecules of interest attached to the protein molecule, can beinduced to bind with a voltage-biased nanopore. The protein shuttle canenable monitoring of the molecules of interest. However, nanopore-basedstudy of molecules is challenging due to the complex geometrical andelectrical charge structures of protein molecules in protein shuttles.The complex structures make it difficult to stably trap a proteinmolecule in a nanopore. Additionally, artificially reproduced nanoporesand nanopore-holding structures typically encounter difficulties withthe sizing of the nanopore, a stability of the bond between the nanoporeand the protein molecule, noise characteristics, and other chemical,mechanical, electrical, and thermal constraints.

To effectively monitor the protein molecules or other molecules ofinterest at a nanopore, systems and methods are needed to induce andmaintain trapping of the molecules at the nanopore. Effective analysisand visualization techniques are also needed to analyze large amounts ofglobal molecule events to reveal the dynamics of a protein shuttlesystem and/or molecules of interest.

SUMMARY

The various examples of the present disclosure are directed towards amethod for characterizing shuttle capture in a nanopore sensor. Themethod can comprise collecting a plurality of time-dependent currentblockage signatures for a plurality of different bias voltages. Themethod can then comprise classifying the plurality of time-dependentcurrent blockage signatures into permanent event signatures andtransient event signatures. For each of the plurality of bias voltages,a protein dynamics landscape (PDL) can be generated for the transientevent signatures. The PDL comprises a collection of blockage spectra.Each of the collection of blockage spectra can comprise a histogram ofblockage values for transient events of a selected maximum duration. Themethod can further comprise, generating for each of the plurality ofbias voltages, a protein dynamics landscape (PDL) for the permanentevent signatures. The PDL comprises a collection of blockage spectra.Each of the collection of blockage spectra can comprise a histogram ofblockage values for permanent events where these blockage values areobtained for each permanent event from the start of the event and up toa maximum time after event start. The method can then compriseidentifying a permanent level blockage value based on permanent eventsignatures. The permanent blockage level is a blockage level that, oncereached, rarely ever changes unless the shuttle is ejected, for example,by reversing the polarity of the applied voltage. The method can furthercomprise identifying an entrance level blockage value based on permanentevent signatures and the PDL plots for the permanent events. The methodcan then comprise selecting one of the plurality of bias voltagescorresponding to a PDL for the transient event signatures. Thecollection of blockage spectra can reveal peaks at blockage values thatare substantially equal to the permanent level blockage value and/or tothe entrance level blockage value.

In some examples, the time-dependent current blockage signaturecomprises a measurement of nanopore current as a function of time duringa shuttle capture event.

In some examples, the classifying can comprise identifying as transientevent signatures any one of the plurality of time-dependent currentblockage signatures which shows a return to an initial current level.The classifying can comprise identifying as the permanent eventsignatures any one of the plurality of time-dependent current blockagesignatures failing to show a return to an initial current level.

In some examples, the method can further comprise calculating a ratio ofa number of permanent shuttle capture events to a number of transientshuttle capture events for each of the plurality of bias voltages. Basedon the calculated ratios, the method can then comprise determining athreshold bias voltage to achieve a preferred ratio.

In some examples, the blockage spectra can comprise an average ofnanopore current blockage data sampled over a fixed time interval toyield histogram data points. The fixed time interval can be at least aslarge as an interval for sampling the nanopore current blockage data.The nanopore current blockage data can comprise a ratio of a change inthe current from an input current to the input current.

In some examples, the identifying step can comprise generating anadditional PDL for the permanent event signatures for at least one ofthe plurality of bias voltages. The identifying step can furthercomprise selecting an entrance level blockage value to correspond to ablockage level lower than the permanent blockage level.

In some examples, the method can further comprise fitting each spectrumof the collection of blockage spectra within a PDL with a sum ofGaussian distributions. The entrance level blockage value can bedetermined from a peak in the collection of blockage spectra within thePDL for permanent events.

In some examples of the first embodiment, the selected maximum durationof the events included in generating particular blockage spectra of aPDL for transient events can be 2 milliseconds, 5 milliseconds 10milliseconds, 30 milliseconds, 100 milliseconds, 500 milliseconds, and1000 milliseconds.

In a second embodiment of the present disclosure, a method can beprovided for characterizing shuttle capture in a nanopore sensor. Themethod can comprise first collecting a plurality of time-dependentcurrent blockage signatures for a selected bias voltage. The method canthen provide for calculating, based on the plurality of time-dependentcurrent blockage signatures, a duration of a shuttle capture event and ablockage percentage or an average blockage percentage for the shuttlecapture event. The method can then provide for determining a blockagelevel based on a distribution characteristic of the plurality oftime-dependent current blockage signatures.

In some examples, the determining step can comprise determining whetherthe duration of the shuttle capture event falls in a range between apre-defined minimum duration and a pre-defined maximum duration.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 2 milliseconds. This can provide ablockage peak in the blockage spectrum for shuttle capture events inthis category at 40% current blockage.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 5 milliseconds. This can provideadditional blockage peaks in the blockage spectrum at 45% and 52%blockages.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 10 milliseconds. This can provideblockage peaks at 40%, 45%, and 52% blockages.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 30 milliseconds. This can provide anadditional blockage peak at a blockage level of 57%.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 100 milliseconds. This can provideblockage levels at 40%, 45%, 52%, and 57%.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 500 milliseconds. This can provideblockage levels at 40%, 45%, 52%, and 57%.

The pre-defined minimum duration can be 0 milliseconds and thepre-defined maximum duration can be 1000 milliseconds. This can providean additional blockage level at 80%.

In some examples, the method can identify an expected protein moleculeorientation based on at least one of a comparison of the PDLs fortransient and permanent events and a presence or absence of blockagepeaks for individual spectra within the PDLs for transient and permanentevents.

In some examples, the determining step can comprise identifying aparticular blockage event as permanent if the duration of the particularblockage event is larger than a threshold time. The threshold time canbe any time value larger than 500 milliseconds in one implementation.

The term “protein shuttle” is used herein interchangeably with the term“molecular shuttle.”

In the present disclosure, the term “shuttle capture” refers to when atleast one molecule is trapped at a nanopore.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings exemplify the embodiments of the presentinvention and, together with the description, serve to explain andillustrate principles of the invention. The drawings are intended toillustrate major features of the exemplary embodiments in a diagrammaticmanner. The drawings are not intended to depict every feature of actualembodiments nor relative dimensions of the depicted elements, and arenot drawn to scale.

FIG. 1A shows an exemplary ClyA nanopore.

FIG. 1B shows a first perspective view of the protein Avidin.

FIG. 1C shows a second perspective view of the protein Avidin.

FIG. 1D shows a third perspective view of the protein Avidin.

FIG. 1E shows a cutout view of an exemplary Avidin trapped in a ClyAnanopore, according to an embodiment of the present disclosure.

FIG. 1F shows a cutout view of an exemplary protein shuttle trapped in aClyA nanopore, according to an embodiment of the present disclosure.

FIG. 2A shows an exemplary nanopore sensor system, according to anembodiment of the present disclosure.

FIG. 2B shows an exemplary set-up for measuring the geometriccontribution to nanopore conductance, according to a scaled upmacroscopic version of the nanopore structure, for example with a 3Dprinted nanopore in plastic material.

FIG. 3A shows an exemplary methodology for using a nanopore sensor,according to an embodiment of the present disclosure.

FIG. 3B shows an exemplary methodology for trapping and releasing aprotein shuttle, according to an embodiment of the present disclosure.

FIG. 4 shows the structure of an exemplary target molecule according toan embodiment of the present disclosure.

FIGS. 5A-5B show an exemplary nanopore and protein molecule structure,according to an embodiment of the present disclosure.

FIG. 6A shows an exemplary methodology for characterizing shuttlecapture in a nanopore sensor.

FIG. 6B shows an exemplary methodology for determining blockage levelsof at least one nanopore in the nanopore sensor.

FIG. 7A-7E show exemplary time-dependent current blockage signatures fortransient events, according to an embodiment of the present disclosure.

FIGS. 8A-8B show exemplary time-dependent current blockage signaturesfor permanent events, according to an embodiment of the presentdisclosure.

FIGS. 9A-9D show a comparison of exemplary nanopore current data ascompared between transient and permanent events, according to anembodiment of the present disclosure.

FIGS. 10A-10F show exemplary PDL histogram plots, according to anembodiment of the present disclosure.

FIGS. 11A-11E demonstrate various exemplary protein moleculeorientations, according to various embodiments of the presentdisclosure.

FIG. 12 shows an X-Y plot of a ratio of permanent events to transientevents as a function of voltage.

FIG. 13 shows a comparison of change in current blockage for Avidinversus an Avidin biotin complex, according to an exemplary embodiment ofthe present disclosure.

FIGS. 14A-14G show exemplary Gaussian fits to the PDL histogram plots,according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

The present invention is described with reference to the attachedfigures, wherein like reference numerals are used throughout the figuresto designate similar or equivalent elements. The figures are not drawnto scale and they are provided merely to illustrate the instantinvention. Several aspects of the invention are described below withreference to example applications for illustration. It should beunderstood that numerous specific details, relationships, and methodsare set forth to provide a full understanding of the invention. Onehaving ordinary skill in the relevant art, however, will readilyrecognize that the invention can be practiced without one or more of thespecific details or with other methods. In other instances, well-knownstructures or operations are not shown in detail to avoid obscuring theinvention. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

The present disclosure is directed to a method for characterizingshuttle capture events in a nanopore sensor. The method first collects aplurality of time-dependent current blockage signatures for a pluralityof different bias voltages. These time-dependent current blockagesignatures can show whether a particular shuttle capture event is apermanent or transient event. The method can then generate a proteindynamics landscape (PDL) for the transient event signatures. The PDLcomprises a histogram of nanopore current data and characterizes currentthrough the nanopore during shuttle capture events. The method can thencomprise identifying an entrance level blockage value based on thepermanent event signatures. This entrance level blockage value canindicate the minimum necessary blockage that a protein shuttle needs tohave when entering a nanopore in order for the nanopore to permanentlycapture the protein shuttle. This information can then be used to selecta particular voltage to cause that particular entrance level blockagevalue.

Therefore, the present disclosure provides a detailed analysis method tocharacterize a nanopore sensor, including levels of blockage of aprotein molecule and how the sensor interacts with different voltages.The analysis can guide interaction with the nanopore sensor to selectcertain voltages in order to obtain corresponding ratios of permanent totransient event captures and even to obtain certain configurations of aprotein shuttle's entry into the nanopore.

The present disclosure provides systems and methods for observing andmeasuring individual, electrically-charged biological molecules as theytranslocate through a voltage-biased nanopore in a lipid membrane bymonitoring the ionic conductance of the nanopore during the molecularmotion through the nanopore. These measurements provide new insightsinto the biophysics of these molecules and their interactions withnanopores. An embodiment of the present disclosure can be used inpractical, portable instruments for sequencing DNA, when a DNA strand isattached to the protein shuttle as the target molecule. Therefore, thepresent disclosure provides for probing the molecule of interest linkedto the shuttle, and probing the dynamics of the molecule of interestlinked to the shuttle.

FIG. 1A shows the protein structure an exemplary nanopore 110 with aninlet diameter 112, an inner lumen diameter 114, an outlet diameter 116,and a length 118.

An exemplary nanopore 110 can comprise ClyA dodecamer. ClyA is apore-forming cytolytic toxin expressed in several pathogenic strains ofEscherichia coli and Salmonella enterica. ClyA has 12 soluble monomers.Upon reaching a target-cell membrane, the soluble monomers undergosignificant conformational changes, involving half of the monomers'amino acid residues, and subsequently assemble into membrane-boundoligomers. The resulting membrane-spanning ClyA pores formnanometer-sized circular holes in the membrane. FIG. 1A shows anexemplary cross-sectional view of ClyA after the ClyA has configured asa pore. The cross-sectional view reveals the twelve-fold rotationalsymmetry axis of the ClyA nanopore after the ClyA nanopore formed acircular hole. Once ClyA configures as a pore in a lipid membrane of acell, the lipid membrane's function as a cell barrier is lost, and thisultimately leads to cell death. The nanometer-sized circular hole isreferred to herein as a nanopore.

A ClyA nanopore can have different diameters for different interiorportions of the nanopore. For example, as shown in FIG. 1A, the inletdiameter 112 of nanopore 110 can be a different width than the innerlumen diameter 114 and both can be different than the outlet diameter116. In some examples, the inner lumen diameter 114 can be smaller thanthe inlet diameter. Further, the outlet diameter 116 can be smaller thanboth the inlet diameter 112 and the inner lumen diameter 114. Ingeneral, the inner lumen diameter 114 is not constant along the lengthof the lumen. For example, the ClyA inner lumen diameter can range from4.1 nm outlet diameter 116 to 6.5 nanometers (nm) inlet diameter 112.Additionally, an exemplary inner lumen diameter 114 can be 5.4 nm. TheClyA nanopore 112 can have an open-pore conductance when configured in alipid membrane or other support structures.

Although the ClyA dodecamer is shown and referred to in the presentapplication as an example nanopore, many different oligomers of ClyAexist. The ClyA 8′mer, ClyA 13′mer, ClyA 14′mer, the tetramer ClyA, andother oligomers can also be employed. Each oligomeric form can havedifferent diameters 112, 114, and 116 and can have a unique open-poreconductance due to its geometric size and the charges of the monomers inthe particular nanopore. The features of a nanopore in FIG. 1A andreferred to subsequently in the present disclosure are not thereforelimited to a particular ClyA oligomer or to ClyA specifically. Anynanopore can be used so long as the nanopore can trap a molecule ofinterest, including a protein shuttle.

FIGS. 1B-1D show various perspective views of a protein 120 with a firstdimension 122, a second dimension 124, a third dimension 126, and afourth dimension 128. For example, FIG. 1B shows a first orientation,FIG. 1C shows a second orientation, and FIG. 1D shows a thirdorientation. Protein 120 can comprise Avidin, which is commerciallyavailable, e.g., Pierce Avidin, ThermoFisher Scientific, and can bepurified from hen egg white.

FIGS. 1B-1D show how Avidin is usually a glyco-protein tetramer whichfurther comprises four beta barrels 123, which are ribbon-likestructures. Selected orientations of Avidin can have an open beta barrel123 open at each end of Avidin. Avidin can also be monomeric, dimeric,and trimeric, and all of these forms can be employed as describedherein. Avidin can comprise one asparagine glycosylation binding site121 per monomer. Avidin has a polysaccharide attached to each of thefour binding sites 121. In addition to the core GlcNAc, thepolysaccharides have 4-5 mannose and 2 GlcNAc. Avidin has an electricalcharge of roughly 7 positive charges at the pH of 7.5. In otherimplementations, deglycosylated avidin can be used.

The positive charge of Avidin allows Avidin to bind with a negativelycharged molecule. Additionally, the dimensions 122, 124, 126, and 128 ofthe protein molecule 120 can be paired to match interior diameters of ananopore such that the protein molecule 120 can fit within a nanopore.For example, a first dimension 122 can be 6.8 nm, a second dimension 124can be 5.5 nm, a third dimension 126 can be 6.7 nm, and a fourthdimension 128 can be 7.1 nm.

Although Avidin is referenced as an exemplary protein molecule forpurposes of the present application, any protein molecule can be used solong as the protein molecule can attach to a nanopore and block at leastsome of the current through the nanopore.

FIG. 1E is a cross-sectional schematic view of the nanopore 110 of FIG.1A with a protein 120 sited in the nanopore 110. In the variousembodiments, the protein's cross-sectional extent can be greater thanthe smallest inner diameter of the nanopore; i.e., the proteincross-sectional extent is at least as large as the smallest nanoporediameter along nanopore length. Further, the protein's cross-sectionalextent can also be greater than the extent of the inner nanoporediameter at sites along the nanopore length other than the site of thesmallest inner diameter.

For example, referring to FIG. 1C, the cross-sectional extent of theprotein Avidin is larger than both the smallest ClyA inner diameter, 4.1nm, and the mid-section diameter of the ClyA nanopore, having an innerdiameter of 5.4 nm. Even with this larger radial diameter, an exemplaryprotein 120 can enter the nanopore 110 by virtue of, e.g., mechanicalflexibility in the structure of protein 120. In addition, one or bothends of nanopore 110 can have an inner diameter that is greater than anextent of protein 120 to thereby enable entry of protein 120 at leastpartially into nanopore 110 if protein 120 cannot otherwise enternanopore 110. With this arrangement, protein 120 can enter nanopore 110and snugly fit within nanopore 110 at one or more sites along the lengthof nanopore 110. This results in match between a nanopore 110 and aprotein 120, such as the case for a ClyA nanopore and an Avidin protein.

In some examples, Avidin can be used to build a protein shuttle whichcan be trapped by a ClyA dodecamer nanopore. For the protein shuttle,Avidin can be configured as the protein 120 that enters nanopore 110, asshown by FIG. 1E. The protein shuttle can have additional moleculesattached to the protein shuttle, as shown further with regards to FIG.1F.

Referring back to FIG. 1E, a cross-sectional extent of protein 120 canbe substantially similar in size to an inner lumen diameter of thenanopore 110. For example, the cross-sectional extent can be 5.5 nm andthe inner lumen diameter can be 5.4 nm. Avidin-ClyA is an exampleprotein-nanopore pair that demonstrates a radial geometry in whichprotein 120 has a cross-sectional extent that is at least about thediameter of nanopore 110 or slightly larger than the diameter ofnanopore 110 at one or more sites along the nanopore length. Further,the ClyA nanopore includes an inlet diameter of 6.5 nm that is largerthan at least the cross-sectional extent of the Avidin protein, wherebythe Avidin protein can enter the ClyA nanopore and then snugly fit at asite within the nanopore along the nanopore length.

Additionally, an inner lumen diameter of nanopore 110 can prohibitprotein 120 from entering nanopore 110 in certain configurations. Forexample, Avidin can be oriented so that the fourth dimension the fourthdimension 128 (7.8 nm) enters the inlet of nanopore 110. Avidin's fourthdimension is much larger than the ClyA inlet diameter 114 of FIG. 1A(6.5 nm). If Avidin entered the ClyA nanopore in an orientation wherethe fourth dimension 128 extends along the inlet diameter 114, thenAvidin would not fully enter the ClyA nanopore. Therefore, FIG. 1E showshow a match in dimension between a nanopore diameter and a proteincross-sectional extent can enable docking in a particular orientationand or location at or in the nanopore. In addition, or as analternative, electrical charges in the nanopore lumen and/or on theprotein can enable docking in a particular orientation and/or locationat or in the nanopore (not shown).

The fitting of Avidin in a ClyA12 nanopore is a close fit and can createa stable bond. The stable bond enables the study of any target moleculeattached to the Avidin. FIG. 1E shows molecular models, with dimensions,of the protein Avidin and of the dodecamer ClyA12 nanopore. The presentapplication leverages this close match between the cross-sectionalextent of Avidin and the inner ClyA12 nanopore lumen diameter, forvarious well-defined Avidin orientations and at various points along thelength of the ClyA12 nanopore. As a result, this pair is a particularlyviable embodiment as an arrangement for a nanopore-matched proteinshuttle as provided herein. In some cases, deglycolitaed avidin can beused as a protein shuttle.

Although Avidin with the ClyA nanopore is discussed within the presentapplication, any other protein shuttle and nanopore combination can beused, where the cross-sectional extent of the protein shuttle is atleast about the extent of the smallest diameter of the inner lumen of ananopore. In one example, a smallest diameter of the nanopore lumen isno greater than the cross-sectional extent of a protein shuttle fittedin the nanopore lumen. In another implementation, a protein shuttle withdimensions smaller than the pore lumen diameters can also be used aslong as the minimum lumen diameter is smaller than the cross-sectionalextent of the protein shuttle and as long as the protein shuttle can bestably trapped at a particular location and with a particularorientation relative to the nanopore.

FIG. 1F is a cross-sectional schematic view of the nanopore 110 andprotein molecule 120 including a target molecule 160 appended to theprotein molecule 120 in the nanopore 110 by way of a primary linkingspecies 140 and a secondary linking species 150 which are attachedbetween the target molecule 160 and the protein molecule 120 in thenanopore 110. This protein shuttle enables nanopore-based study of thetarget molecule 160 by drawing the target molecule 160 to and/or atleast partially into the nanopore 110 by means of the protein molecule120. The protein shuttle can have more or fewer components as shown inFIG. 1F, so long as the protein shuttle has a protein molecule 120.

The primary linking species 140 can connect the protein molecule 120 tothe secondary linking species 150. The primary linking species 140 canalso connect the secondary linking species 150 to the target molecule160. In some examples, there can be multiple chains of a proteinmolecule 120 connected to a primary linking species 140, a secondarylinking species 150, a primary linking species 140 and another proteinmolecule 120. However, more than one protein molecule 120 is not needed.

In some examples, protein molecule 120 can be positively charged andconfigured to attach to a negatively charged nanopore 110. For example,ClyA is negatively charged along an interior ring.

A judicially chosen protein-pore pair can serve as a platform for thestudy of protein 120, through deterministic clearing of protein 120 fromnanopore 110 with a control voltage, as discussed further below.Referring back to FIG. 1F, there can also be studied a wide range oftarget molecules 160 and their interaction with analytes in solution(not shown). A target molecule 160 that is linked to the protein shuttleby way of primary 140 or secondary 150 linking species can be drawn toand studied at nanopore 110 by virtue of its linking to the proteinshuttle. As explained above, the protein shuttle can be matched to thesizing of a particular nanopore 110. Properties of the protein shuttle,such as electrical charge, can be exploited to attract the proteinshuttle to nanopore 110. The nanopore with matched protein shuttleenables studies of both the protein shuttle and target molecule 160.Other molecules can be linked to the protein shuttle or can react withor at the target molecule, and these molecules can be studied viananopore 110 as well. For example, substrates and products of a reactioncatalyzed by target molecule 160 can be studied due to a secureattachment between protein molecule 120 and nanopore 110.

The protein shuttle can have one or more characteristics that enableprotein molecule 120 to be driven to and/or driven at least partiallyinto selected nanopore 110 along with a target molecule 160. Forexample, the protein Avidin has a significant positive electrical chargeat the pH of the native environment of most target molecule proteins. Asa result, for the study of target molecule proteins, Avidin can beelectrophoretically, electro-osmotically, or otherwise be electricallyattracted to and drawn into nanopore 110 with target molecule 160 linkedto the Avidin in the molecular shuttle configuration for study of targetmolecule 160 at nanopore 110.

In some examples of the present disclosure, protein molecule 120 can beAvidin and primary linking species 140 can be biotin. FIGS. 1B-1C showedthat each beta barrel 123 of Avidin is open at one end. This allows aprimary linking species 140 to enter and bind to residues inside betabarrel 123. Referring back to FIG. 1F, biotin can be an exemplarylinking species configured to bind to residues inside beta barrels 123of Avidin. The binding of biotin to Avidin represents the strongestnon-covalent ligand bond found in nature, with a dissociation constantof 10-15 M. The strong bonding between the protein Avidin and its ligandbiotin can be exploited to enable the attachment of a linking species toAvidin by way of biotin bonding to Avidin, so that target molecule 160can be configured with secondary linking species 150 in a molecularshuttle arrangement for study of target molecule 160 at nanopore 110.Biotin has strong and specific affinity to Avidin. Biotin can bind in abiotin binding pocket of avidin. Avidin has one binding pocket permonomer. Since Avidin is a tetramer, there are four binding pockets peravidin.

Linking species 140 can include molecules with biotin at one location.For example, the linking species 140 can be a linear molecule that has anumber of polyethylene glycol (PEG) units to adjust the length. Biotincan be at one end and maleimide can be at the other. For example, biotincan bind to Avidin on one end. Maleimide can bind to a naturallyoccurring or an engineered-in cysteine residue on a target molecule 160at the other. The secondary linking species might not be needed.

The secondary linking species 150 can be provided as, e.g., a polymermolecule, a protein, DNA, Cas9, lysine, a peptide, or other suitablespecies that can be attached to a target molecule 160. The secondarylinking species 150 attached to the Avidin through biotin bonding to theAvidin and secondary linking species. The biotinylation of secondarylinking species 150 thereby enables a strong bond between anAvidin-biotin complex and secondary linking species 150. Attachment ofsecondary linking species 150 to target molecule 160 provides anAvidin-biotin-linking species-target molecule arrangement for study oftarget molecule 160 by nanopore 110.

In some examples, target molecule 160 can comprise a DNA sequence, anRNA sequence, Cas9, or any other molecule for studying. In someexamples, there can be more than one target molecule 160. In someexamples, target molecule 160 can be any part of the shuttle, includingprotein molecule 120 or primary 140 or secondary 150 linking species.For example, a protein shuttle, according to an embodiment of thepresent disclosure, can be used to shuttle Cas9. Cas9 can be introducedto a primary and or secondary linker and then to Avidin, andsubsequently captured by a nanopore. Once the Cas9 is captured, it canbe used to do CRISPR analysis.

A protein shuttle can include a number of primary 140 and secondary 150linking species that are themselves proteins, e.g., neutrAvidin,streptAvidin, or other suitable species, which can be exploited todeterministically orient an attached target molecule 160.

In some examples, target molecule 160 can comprise a DNA sequence, anRNA sequence, Cas9, or any other molecule for studying. In someexamples, there can be more than one target molecule 160. In someexamples, target molecule 160 can be any part of the shuttle, includingprotein molecule 120 or primary 140 or secondary 150 linking species.

Given the deterministic orientation of the shuttle (for example avidin)in the nanopore when the shuttle (for example avidin) is captured in itsstable trap state (the docked state), the orientation of the targetmolecule 160 will also be deterministic if the point of linkage to thetarget protein is known (for example when a cystine is engineered in tothe target protein in a particular residue location) and the length ofthe linker is chosen appropriately for that target molecule. In someexamples, primary 140 and/or secondary 150 linking species can have aform fitting arrangement in nanopore 110. Therefore, primary 140 and/orsecondary 150 linking species can orient the protein shuttle in nanopore110 in a deterministic arrangement that extends to target molecule 160.Thereby, target molecule 160 is provided with a deterministic directionrelative to nanopore 110. In one example of such, between one and fourAvidin linkers are employed for the tetramer Avidin, given that such asfour binding sites for biotin. In this protein shuttle arrangement, onlyone of the species, e.g., the leading Avidin, needs to provideproperties that enable driving of the shuttle to nanopore 110. Forexample, only the leading Avidin needs to be electrically charged toenable electrophoretic or electro-osmotic driving of the shuttle. Thelinking Avidin species can be neutrAvidin or streptAvidin. Otherprotein, and other linking species can be employed.

For any of the protein shuttle arrangements described above, there canbe shuttled any suitable target molecule 160, including a PSII molecule.PRI is an enzyme that catalyzes the splitting of water molecules, intofour protons, four electrons, and one oxygen molecule per two watermolecules. The conformational dynamics and changes during the four-stepcatalytic process can be studied. The process is driven by sunlight(photons) so in this case the reagents (substrates) for the enzymaticprocess are two water molecules and 4 photons, and the products are fourprotons, four electons, and one oxygen molecule.

The shuttle protein can be stably linked to target molecule 160. Thelinking species 140 can be specific to particular location(s) on theprotein molecule 120 and on the target molecule 160. The linking species140 can have a variable length to match the needs for a specific targetmolecule. For example, with respect to PEG units, the length of thelinking species 140 is adjusted with the number of PEG units betweenbiotin and maleimide. The protein molecule 120 can be linked to a broadclass of important target molecules 160. Proteins, including enzymes,can be linked as target molecules 160 with appropriate linkers, forexample the biotin—PEG units—maleimide linker.

Any suitable molecule can be studied as the target molecule.

FIG. 2A shows an exemplary nanopore sensor system 200A, according to anembodiment of the present disclosure. The nanopore sensor system 200Acan include a first electrode 210A, a second electrode 210B, a firstfluidic chamber 212, a second fluidic chamber 214, a protein molecule216, a nanopore 218, support structure 220, a current 222, a currentsensor 224, a linking species 226, a target molecule 228, and biotin230. Although not shown in FIG. 2A, system 200A can include a flushingsystem whereby molecules can be flushed on demand separately from thefirst and second fluidic chamber. In some cases, this system can also beused to inject molecules into the first and or second fluidic chamber.

The first electrode 210A can be disposed in the first fluidic chamber212. The second electrode 210B can be disposed in the second fluidicchamber 214. Electrodes 210A, 210B can be configured to apply a voltagebetween chambers 212, 214. Fluidic chambers 212, 214 can have fluids inthem. For example, second fluidic chamber 214 can have a fluid with anegative voltage relative to a fluid in first fluidic chamber 212. Thesupport structure 220 can comprise a lipid bilayer or other materialconfigured to trigger fluidically separate chambers 212, 214.

An applied voltage bias polarity can generate a current 222 which can beset based on the electrical charge of a selected protein 216 to causethe protein to be electrically driven into nanopore 218 from one of thechambers. For example, for drawing Avidin into a nanopore, secondfluidic chamber 214 is kept at negative voltage relative to firstfluidic chamber 212, and Avidin is provided in first fluidic chamber212. Therefore, characteristics of protein shuttle 216, e.g., electricalcharge and or electric dipole moment, enable voltage-controlled docking,undocking, capture, and uncapture of protein shuttle 216.

After docking of just the charged protein 216 (avidin for example, moregenerally the shuttle without target protein,) in or at the pore, therecan be non-zero residual current through the pore and that residualcurrent should have low noise background. For example, theroot-mean-square current noise for a 1 kHz bandwidth can be 10% or lessof the residual current. An Avidin dimer or an avidin monomor can alsobe used as a shuttle in combination with a nanopore, for example, thenanopore can be ClyA or Mycobacterium smegmatis porin A (MSPA).

Turning to further specifics of the system 200A, a nanopore 218 can beprovided in support structure 220 in any suitable manner. For example,ClyA nanopores can be provided in a lipid bilayer by adding ClyA tofirst fluidic chamber 212 of the system 200A. When a single one of theClyA nanopores inserts itself into the membrane, as shown in 200A, thenionic current 222 measured in current sensor 224 of 200A increases dueto ionic current flow through the nanopore between the two chambers ofsolution. Once nanopore 218 is in place in support structure 220,protein 216, or protein-based molecular shuttle can beelectrophoretically or otherwise driven into nanopore 218. Prior to thisstep, nanopores remaining in solution can be flushed from first fluidicchamber 212.

In some examples of the present disclosure, the nanopore sensor system200A can include particular components. The support structure 220 can bea lipid bilayer membrane suspended across a 40 micrometer Teflon framein an electrolytic buffer solution (150 mM NaCl, 15 mM TRIS pH 7.5). Asilver/silver-chloride electrode 210A, 210B can be placed in eachreservoir and a current 222 of a few tens of mV can be applied acrossthe membrane. The membrane electrically isolates the two reservoirs, andinitially no current 222 flows between the electrodes 210A and 210B.Preformed ClyA pores can be added to the first fluidic chamber 212, andwhen a single pore 110 inserts in the membrane—observed as a step changein current to a stable open-pore current—the first fluidic chamber 212is immediately flushed with buffer electrolyte solution to preventadditional ClyA pores from inserting into the membrane. Once a singlepore is in place in the membrane, 10 pmol of Avidin can be added to thefirst fluidic chamber 212. The current 222 is transiently blocked whenindividual, positively charged Avidin molecules are trapped by andsubsequently escape or are ejected from the nanopore 218.

The ionic conductance through an individual voltage-biased nanopore 218can be measured and analyzed prior to protein 216 insertion in the firstfluidic chamber 212. For the exemplary ClyA nanopore, a conductance of1.66 nS, measured at a 30 mV bias, is found to be common and can beemployed for the capture of Avidin. ClyA nanopores with a conductancewithin a percent of this value are the most common ones observed asinserting in a membrane and are the most stable over time.

In one example of a method for driving a protein into a nanopore, Avidinis inserted in a ClyA nanopore. For example, after 10 pmol (1 microlitervolume) of Avidin is added to the 250 microliter first fluidic chamber212 of 200A with a 1.66 nS ClyA nanopore in place on a lipid bilayer,the current through the −35 mV voltage-biased nanopore is observed totransiently drop from an open-nanopore value of about 58 pA asindividual Avidin protein molecules are captured by, and escape from,the nanopore 218. In some cases a permanent trap state of avidin in ClyAcan be observed: this can be referred to as the docking state of theshuttle protein avidin in ClyA. For measuring current 222 there can beemployed any suitable current sensor 224, e.g., a Molecular DevicesAxopatch patch clamp amplifier. The output signal can be processed,e.g., by a 10 kHz, 4 pole Bessel filter, to minimize high frequencycurrent noise, and then sampled and digitized, and stored, forprocessing.

The linking species 226 can be connected to the protein molecule 216 andthe target molecule 228 via biotin 230. The target molecule 228 can be aPSII target molecule.

The Avidin that is inserted into a nanopore 218 in system 200A can bearranged as a protein shuttle, including one or more linking species 226and target molecules 228. The protein molecule 216 and target molecule228 in the chambers 212 and 214 of a nanopore system 200A, the Avidin orother lead protein can be drawn to and trapped in the nanopore 218,e.g., the ClyA nanopore, from the first fluidic chamber 212.

The shuttle structure with protein molecule, target molecule, andlinker(s) can also be pre-formed before injection of the shuttlestructure units into first or second fluidic chamber.

In an alternative configuration, the Avidin, or other leading structureof the shuttle, is disposed in one of the two fluidic chambers, e.g.,first fluidic chamber 212, and target molecule is disposed in theopposite of the two fluidic chambers, e.g., second fluidic chamber 214.The target molecule 228 here can be, e.g., biotinylated, with linkingspecies 226 and biotin 230 at the end of linking species 226 oppositetarget molecule 228. The target molecule 228 can arrive at nanopore 218from second fluidic chamber 214, and link to protein 216 that is alreadytrapped in nanopore 218, through an outlet of the nanopore to secondfluidic chamber 214. After this linking has occurred, the voltage orother stimulus for driving protein 216 to nanopore 218 can be adjusted,e.g., to lessen the trapping of protein 216, drawing protein 216 backtoward first fluidic chamber 212 and thereby pulling target molecule 228further to nanopore 218 from second fluidic chamber 214, via linkingspecies 226 that is attached between protein 216 and target molecule228.

System 200A thus can provide for electrophoretic driving and capture ofAvidin 216 and correspondingly, of a molecular shuttle including of anAvidin-biotin+linking species+target molecule arrangement, into ananopore 218. System 200A provides for deterministic control of anAvidin protein shuttle attached via biotin 230 and a linking species 226to a target molecule 228, here a PSII photosynthetic enzyme, forcontrollable direction and orientation of the PSII target molecule 228toward and at least partially into the nanopore.

Protein molecule 216 can be trapped in nanopore 218 and target molecule228 can partially enter nanopore 218 from first fluidic chamber 212. Thecapture of protein molecule 216 by nanopore 218 in the docking state andwith the target molecule linked to the protein molecule can beconsidered a blockage event. A change in current signal in thissituation from the current signal when the protein molecule 216 alone isdocked, when the same applied voltage between the fluidic chambers isapplied in the two cases, is the current signal that is of interest andcontains the signals corresponding to target molecule sensing with thesensor platform. In some examples of the present disclosure, targetmolecule 228 can be disposed in the second fluidic chamber 214 and canbe captured by nanopore 218 such that target molecule 228 at leastpartially obstructs the at least one nanopore during a blockage event(not shown).

FIG. 2A shows a nanopore system 200A where a protein 216 such as Avidinthat is provided in first fluidic chamber 212 can beelectrophoretically, electro-osmotically, or otherwise driven tonanopore 218, at least partially taken into nanopore 218, held atnanopore 218 for a selected duration of time, and then subsequentlyreleased from nanopore 218 back into first fluidic chamber 212. Thereleased protein 216 can then be re-drawn to and re-trapped in nanopore218. Alternatively, a different protein from first fluidic chamber 212,or from second fluidic chamber 214, can be driven to nanopore 218 fortrapping. When arranged in a molecular shuttle, the Avidin thereby canbe controlled as the engine that moves the shuttle toward, into, and outof nanopore 218. Thereby, target molecule 228 linked to protein 216 canbe captured at nanopore 218, for a controlled capture time, to enableevaluation of linked target molecule 228, even at the single moleculelevel. Depending on the size of target molecule 228, target molecule 228can be pulled fully into nanopore 218 with Avidin, or as shown here, canbe pulled partially into nanopore 218. Target molecule 228 alternativelycan be pulled to the entrance of nanopore 218. The understanding of theunperturbed electronic properties of the Avidin-ClyA nanopore platformas well as the biotin-Avidin-ClyA system provided herein enable suchtarget molecule capture and evaluation.

A matched pair of a nanopore and a charged protein shuttle can cause theshuttle protein to dock in a particular and reproducible orientation ator in the nanopore. For example, a ClyA dodecamer can be matched withAvidin tetramer. This combination is ideal for a broad range of pHvalues. A shape form fit can be used to ensure that the charged proteinshuttle docks in the pore at a particular location and with a particularorientation relative to the pore. For example, Avidin can be caused todock in at a particular orientation leading to a blocking of a currentthrough the nanopore. An applied voltage can cause the protein shuttleto be controllably docked and undocked.

FIG. 2A demonstrates the operation of the protein shuttle, here ashuttle including an Avidin-biotin+linking species+target moleculearrangement, for controllably directing a target molecule to and perhapsinto a nanopore. System 200A allows for trapping, in or at the nanopore,molecules of interest that have low or no net charge, and for trappingmolecules of interest with controlled orientation relative to thenanopore.

FIG. 2B shows an exemplary macroscopic nanopore sensor set-up 200B formeasuring nanopore conductance, according to an embodiment of thepresent disclosure. FIG. 2B includes many of the same components as FIG.2A and further comprises a voltmeter 232, and a transformer 234.Therefore, the nanopore sensor set-up 200B can measure ionic currentflow through nanopore 218 between first fluidic chamber 212 and secondfluidic chamber 214.

For example, a macroscopic nanopore 218 can be glued into a thininsulating plastic sheet to simulate the lipid membrane. The sheet canseparate two reservoirs of 150 mM NaCl in water, and in each reservoirhemispherical electrodes 210A and 210B of 11.4 cm radius surrounded thepore. Conductance measurements on the macroscopic pore system 200B canbe made with a range of 60 Hz AC voltages from 10-50 volts rms.Conductance can be calculated from a linear slope of the I-V plot. Nophase shifts between observed currents and applied voltages should beobserved. A temperature of the electrolyte can be monitored andmeasurements can be restricted to a few seconds in duration to minimizeelectrolyte Joule heating.

Scaling the measured conductance value by 3/107 obtains the geometricalconductance for nanopore 218 at 2.87 nS. The inverse of the measuredgeometric pore conductance is a sum of the inverse conductance for thepore alone, and the access resistance. The correction of accessresistance to infinite electrode radius can increase the totalresistance by 1.57 percent. This measured and scaled conductance valuewill be significantly reduced by exclusion of part of the chlorine ions'contribution to the pore conductance. To evaluate the reduction factor,a 1.66 nS ClyA nanopore can be captured in a lipid bilayer membrane witha 150 mM buffered salt solution on both sides. First fluidic chamber 212can be refilled with a 20 mM buffered salt solution. This can create ameasurable open circuit potential of 26 mV across nanopore 218 due to anunequal transfer of sodium versus chlorine ions across the pore in thedrift-diffusion process.

The Goldman-Hodgkin-Katz (GHK) flux equation from ion-channel biophysicscan analyze the measured open circuit potential across the pore. The GHKequation can calculate the permeability factor reflecting the extent towhich the chloride ions are blocked, relative to the sodium ions, frompassing through nanopore 218. Negative charges in the lumen close to theoutlet aperture can repel negative chloride ions in the NaCl-saltsolution and result in a reduced mobility and diffusion constant forchloride ions passing through the pore. By applying a salt gradientacross the pore, this difference in mobility and diffusion constant cancause a voltage across the pore to build up. By measuring this voltagein steady state, i.e., when there is no current through the pore, theGHK equation can deduce the ratio of the mobilities of chloride andsodium ions, and thereby deduce the relative charge exclusion forchloride ions by the pore.

The GHK calculation results in a permeability factor of 0.22, which issmaller than the 0.33 obtained at higher molarities. A larger chlorineion blockage effect should be expected in the macroscopic set-up 200Bdue to a larger Debye screening length at lower ionic strength. Theobtained permeability factor reduces nanopore conductance from thegeometrical pore conductance value. This analysis can help predict aClyAl2 pore conductance under the conditions of the system shown in FIG.2A, where the pore conductance should therefore be 1.75 nS.

Simple scaling arguments show that the conductance prediction for theother possible oligo candidate, the slightly larger 13′mer, would be1.97 nS, which rules it out. A higher conductance shows that thenanopore is not a good fit for the protein molecule. For example, the13′mer pore's larger size (diameter) no longer makes it a formfittingpore for Avidin so Avidin does not dock in the 13′mer pore in aparticular location and with a particular orientation. A small number ofpores exist with 1.9 nS, but these pores are somewhat unstable wheninserted in membrane 220. Therefore, FIG. 2B shows an exemplarymacroscopic system 200B which can identify constraints for appropriatemicroscopic systems (for example, the system of FIG. 2A).

FIG. 3A shows an exemplary methodology 300A for using a nanopore sensor,according to an embodiment of the present disclosure. The exemplarymethodology 300A can use a nanopore sensor as described with respect toFIGS. 2A-2B. The methodology 300A can provide a method of analyzingmolecular trapping in and or at one or more nanopores.

The method can begin at step 310 by applying a voltage across the one ormore nanopores. The voltage can draw at least one protein shuttletowards at least one of the one or more nanopores. The protein shuttlecan comprise an electrically charged protein molecule. In someinstances, the protein shuttle can further comprise one or more linkingspecies, and a target molecule. While applying the voltage, the methodcan provide for measuring an ionic current through at least one of theone or more nanopores in step 312.

While measuring the ionic current, the method can further provide fordetecting a blockage event in step 314 based on the ionic current for atleast one of the one or more nanopores. A blockage event indicates acapture of the protein shuttle by at least one of the one or morenanopores. A blockage event can be detected through a change in a totalionic current flow or a change in an ionic current flow for a particularone of the one or more nanopores. The change in the ionic current flowcan comprise a decrease in the ionic current flow.

In some examples of step 314, the methodology can optionally includemaintaining a voltage after a blockage event had occurred. In otherexamples, step 314 can include reducing the voltage after a blockageevent has occurred. In other examples, step 314 can include increasingthe voltage after a blockage event has occurred. In other examples, 314can include reversing a voltage after a blockage event has occurred.

After detecting a blockage event, the methodology 300A can proceed tostep 316, where a duration is measured for the blockage event. Aftermeasuring the duration, the methodology can further comprise determiningan average ionic current during a blockage event 318.

In some examples, the method 300A can comprise inducing an ejection ofthe captured protein shuttle by reversing a polarity of the voltage. Forexample, the method 300A can comprise automatically reversing thevoltage after some selected duration. For example, the duration can beone second of measured current blockage. This voltage polarity reversalejects the positively-charged protein from the nanopore. The voltagebias can then be returned to a negative value, after which theopen-nanopore current is again observed, followed by new currentblockage capture events. If the protein shuttle with attached targetmolecule was originally provided in the first fluidic chamber, then theshuttle and target molecule are sent back to the first fluidic chamber.In some cases, by increasing the voltage after the shuttle is trapped,the shuttle can be ejected into the second fluidic chamber even thoughit originally entered the pore from the first fluidic chamber.

This method can be used for one or more nanopores, for one or moreprotein shuttles, and/or for one or more blockage events. Therefore, themethodology 300A provides the means to determine whether a proteinshuttle was trapped by a nanopore, according to the example shown inFIG. 2C, for example.

FIG. 3B shows an exemplary methodology 300B for trapping and releasing aprotein shuttle, according to an embodiment of the present disclosure.The exemplary methodology 300B comprises providing a nanopore sensor atstep 322. The nanopore sensor can be according to the embodiments of thepresent disclosure discussed with respect to FIGS. 2A-2C.

Referring back to FIG. 3B, the method 300B can then comprise introducinga protein shuttle at step 324. The protein shuttle can be a proteinshuttle as discussed with respect to FIGS. 1F and 2A. The proteinshuttle can be introduced into a first fluidic chamber of the nanoporesensor. In some examples of the present disclosure, the protein shuttlecan be introduced into a second fluidic chamber of the nanopore sensor.

The method 300B can then comprise applying a voltage to trap the proteinshuttle by a nanopore in step 326. In step 328, the method can providefor inducing an ejection of the protein shuttle from the at least onenanopore by reversing a polarity of the voltage.

Therefore, FIG. 3B provides a methodology 300B for capturing andreleasing a protein shuttle, according to an embodiment of the presentdisclosure.

FIG. 4 shows the structure of an exemplary target molecule 400 accordingto an embodiment of the present disclosure. The exemplary targetmolecule can include lipids or detergent molecules 410; a beta-secretasecleavage site 420; a gamma-secretase cleavage site 430; and a linkermolecule 440. In some examples, the target protein can be APP, amembrane-bound protein. The beta-secretase can bind to and cleave theAPP protein. The linker molecule can link to a protein molecule, such asAvidin. The cleavage enzymes, β-secretase and γ-secretase cansequentially bind to and cleave the APP protein. By trapping the shuttlewith the linked APP protein in the ClyA nanopore, β-secretase and/orγ-secretase can be added to the fluidic chamber of a nanopore sensor.The resulting time-dependent current signals can be monitored to examinethe dynamics of the enzymes binding to and cleaving the APP protein.Therefore, for example, a system using this sort of exemplary target canprobe whether the dynamics of the nanopore sensor system are changedwith targeted drugs aimed at inhibiting the cleavage activity ofβ-secretase or γ-secretase. The APP cutting by β-secretase andγ-secretase is believed to be of importance for Alzheimer's disease asthe cleaving of APP by these enzymes leads to formation of cleavagefragments Aβ that will form β-Amyloid plaques. APP is β-amyloidprecursor protein. Aβ is amyloid β-peptide). γ-secretase includescatalytically competent γ-secretase (for example PS1 containingcompetent γ-secretase complex) and soluble γ-secretase (for example withdetergent enzyme solubilization) including soluble competentγ-secretase. β-secretase includes soluble β-secretase. In FIG. 4 thelinker is schematically shown to be linked to the APP protein in onelocation. Dependent on the linker position—for example the linker canalso be linked to the opposite end of APP—the platform can be chosen formaximum sensitivity to cleaving of APP by β-secretase and/orγ-secretase.

Any suitable target molecule can be studied at a nanopore platform usinga protein shuttle, e.g., an amyloid precursor protein (APP) with lipidsor detergent molecules. FIG. 4 shows an example of a target protein 400structure to be probed, namely, an APP protein, which is amembrane-bound protein, in lipid or with detergent molecules. Thisprotein can be arranged with the protein shuttle for study of theprotein at a nanopore. For example, the dynamics of the workings ofcutting enzymes such as beta or gamma secretase, and the effect of drugtargets on the system, can be studied with the aid of thenanopore-matched protein shuttle, and the shuttle can include any numberof linking species as described above.

FIGS. 5A-5B show an exemplary macroscopic nanopore 510 and proteinmolecule structure 520, according to an embodiment of the presentdisclosure. FIGS. 5A-5B show 3D printed macroscopic models. FIG. 5Ashows an Avidin-nanopore configuration wherein a rigid Avidin molecule520 sits atop a rigid ClyA12 pore 510 oriented to obtain maximum currentblockage of 10% in macroscopic conductivity experiment. FIG. 5B shows anelastic Avidin model 520 maximally pressed into rigid ClyA12 pore model510 achieving 20% blockage in macroscopic conductivity experiment. Scalebar corresponds to 3 nanometers for actual protein and nanopore size.

The nature of the Avidin-ClyA interaction described above for a 1.66 nSClyA nanopore can be understood on a molecular level if the number ofprotomers in the ClyA oligomer is known. To address this issue, anexperimental methodology can determine which oligo is expected to havethe observed conductance of 1.66 nS. There are two phenomena thatdetermine the conductance. One is simply the geometry of the nanopore.This geometric-based conductance is then modified by the presence ofcharged amino acids on the walls of the nanopore. In fact, in ClyA thelimiting site of smallest diameter along nanopore length is known tohave a highly negative charge that can block chlorine ion conductancethrough the nanopore. Geometric and charge selectivity effects can bedetermined in different experiments and assembled to predict the totalconductance of a given nanopore.

To determine the geometrical contribution to the nanopore conductancethere can be constructed a model, e.g., 3D-printed model, of a selectednanopore, such as ClyA, scaled up by a factor of 107/3 to atomiccoordinates for the ClyA nanopore, as-obtained from the proteindatabase. The 3D printer used to prepare the scaled-up nanopore was aFormLabs Form 2 model, with a resolution of ˜50 microns. The dimensionsof the molecules were scaled up by a factor of 107/3 from the PDBdatabase. The plastic used for the rigid models was FormLabs Clear (Part# FLPGCL02, FLPGCL03).

To model a protein 520 fitted in a nanopore 510 as provided herein, a3D-printed Avidin protein 520 can be prepared with the same scaling asused for the ClyA12 3D-printed nanopore 510. The 3D printing plasticused for the flexible Avidin model was FormLabs Flexible (Part #FLFLGRO2) with a Shore Hardness of 80A.

Regardless of orientation, the 3D-printed protein 510 was slightly toolarge to completely enter the 3D-printed ClyA nanopore lumen, mostlybeing blocked from entry by a few residues on the outside surface of theAvidin 520. Placing the molecule on the rim of the pore 510 in themacroscopic conductance experiment, as shown in FIG. 3A, only reducesthe conductance by 10%. With Avidin 520 deeply inserted in the lumen ofthe pore a shown in FIG. 5B, only a 20% conductance reduction wasobserved.

Geometrically, elasticity of the protein and/or the nanopore can allow aprotein to enter into a nanopore more deeply and with more intimatecontact under the force provided by the electric field from the appliedvoltage bias. In addition, electrostatic charges in a nanopore lumen caninteract strongly with electrostatic charges on a protein at shortdistance when the protein 520 is near the bottom of the nanopore 510.And ionic charge-selectivity effects for conductance may become moreintense when a protein 520 is in a nanopore 510, also leading to deepercurrent blockades. Based on these considerations, it is therebypreferred that a selected protein 520 have a radial diameter that is atleast about the same inner lumen diameter of a nanopore 510 in which theprotein 520 is to be fitted.

FIGS. 6A-6B are discussed with respect to a nanopore sensor system asdescribed above with respect to FIGS. 2A-2B. Notably, the nanoporesensor system should include at least one nanopore and at least oneprotein shuttle. FIG. 6A shows an exemplary methodology 600A forcharacterizing shuttle capture in a nanopore sensor.

The methodology 600A can begin at step 610 by collecting time-dependentcurrent blockage signatures for a plurality of different bias voltagesat a nanopore sensor. Each single protein capture, whether transient orpermanent, produces its own unique, time-dependent current blockagesignature that can be uniquely identified. The time-dependent currentblockage signature can comprise a measurement of nanopore current as afunction of time during a shuttle capture event. Example time-dependentcurrent blockage signatures for transient and permanent events are shownin FIGS. 7A-8B.

Referring back to FIG. 6A, the method can then proceed to step 612 andclassify the time-dependent current blockage signatures into permanentevents and transient events. Permanent events can be identified for anyone of the plurality of time-dependent current blockage signaturesfailing to show a return to an initial current level (as shown in FIGS.8A-8B). Transient events can be identified for any one of the pluralityof time-dependent current blockage signatures which shows a return to aninitial current level (as shown in FIGS. 7A-7E).

The method can then provide for generating a protein dynamics landscape(PDL) for the transient event signatures at step 614. These PDLs can begenerated for each of the plurality of bias voltages. The PDLs comprisesa collection of blockage spectra. Each of the collection of blockagespectra can comprise a histogram of blockage values for a selectedduration. In some examples, the blockage spectra can comprise an averageof nanopore current data sampled at a fixed interval to yield histogramdata points. A length of the fixed interval can be at least as long as asampling period of the nanopore current data. For example, the length ofthe fixed interval can be 160 microseconds. The nanopore current datacan comprise a ratio of a change in the current from an input current tothe input current.

In some examples, the selected duration of the particular blockagespectra can be between 0 seconds and 2 microseconds, between 0 secondsand 5 microseconds, between 0 seconds and 10 microseconds, between 0seconds and 30 microseconds, between 0 seconds and 100 microseconds,between 0 seconds and 500 microseconds, or between 0 seconds and 1000microseconds.

PDL plot generation is discussed further with respect to FIGS. 10A-10F.

Referring back to FIG. 6A, the method can then proceed to step 616 toidentify an entrance level blockage value based on the permanent eventsignatures. In some examples, the identifying step can comprisegenerating an additional PDL for the permanent event signatures for atleast one of the plurality of bias voltages. The identifying step canfurther comprise selecting the entrance level blockage value tocorrespond to a common lower peak of the blockage values for thecollection of blockage spectra in the additional PDL. The entrance levelblockage can be selected to correspond to a level that often precedes,within a single trapping event, and is lower than the blockage valueobserved when the shuttle protein is permanently trapped in thenanopore.

The method can then comprise selecting one of the plurality of biasvoltages corresponding to a PDL for the transient event signatures basedon the peak blockage values at step 618. The collection of blockagespectra can provide peak blockage values that are substantially equal tothe entrance level blockage value.

The PDL is generated form a collection of blockage spectra. Thedifferent blockage spectra in a PDL for permanent events can begenerated with inclusion of all permanent events recorded withapplication of a particular voltage between the fluidic chambers. Foreach individual blockage spectrum within a PDL, the histogram isgenerated from current data within only an initial time period of eachevent, from the event start to a maximum time. The maximum time ischosen differently for the different individual blockage spectra withina PDL, for example 2 milliseconds, 5 milliseconds, 10 milliseconds, 30milliseconds, 100 milliseconds, 500 milliseconds, and 1000 milliseconds.

The PDL comprises a collection of blockage spectra. Each of thecollection of blockage spectra can comprise a histogram of blockagevalues for permanent events where these blockage values are obtained foreach permanent event from the start of the event and up to a maximumtime after event start. The method can then comprise identifying apermanent level blockage value based on permanent event signatures. Thepermanent blockage level is a blockage level that, once reached, rarelyever changes unless the shuttle is ejected, for example, by reversingthe polarity of the applied voltage. The method can further compriseidentifying an entrance level blockage value based on permanent eventsignatures and the PDL plots for the permanent events. The method canthen comprise selecting one of the plurality of bias voltagescorresponding to a PDL for the transient event signatures. Thecollection of blockage spectra can reveal peaks at blockage values thatare substantially equal to the permanent level blockage value and/or tothe entrance level blockage value.

In some examples, selecting the entrance blockage level can furthercomprise fitting a first Gaussian distribution to each of the collectionof blockage spectra and fitting a second Gaussian distribution to aparticular blockage spectra. The second Gaussian distribution can besubtracted from the first Gaussian distribution to yield resultantdistribution. The entrance level blockage value can be determined from apeak of the resultant distribution. This method is discussed furtherwith respect to FIGS. 14A-14G.

Referring back to FIG. 6A, the method can optionally include anadditional step of calculating a ratio of a number of permanent shuttlecapture events to a number of transient shuttle capture events for eachof the plurality of bias voltages. Based on the calculated ratios, themethod can then comprise determining a threshold bias voltage to achievea preferred ratio.

FIG. 6B shows an exemplary methodology 600B for determining blockagelevels of at least one nanopore in the nanopore sensor. The method canprovide for first collecting a plurality of time-dependent currentblockage signatures for a selected bias voltage in step 620.

The method can then provide for calculating a duration of a shuttlecapture event and a blockage percentage for the shuttle capture event instep 622. Calculating the duration of the shuttle capture event and theblockage percentage can be based on the collected plurality oftime-dependent current blockage signatures. For example, a change in thenanopore current while a protein molecule is attached relative to thenanopore current while no protein molecule is attached can identify whatpercentage of the original nanopore current is blocked by the shuttlecapture event. A duration can be identified from a length of time of theshuttle capture event.

The method can then comprise determining a blockage level based on adistribution characteristic of the plurality of time-dependent currentblockage signatures at step 624. In some examples, step 624 can comprisedetermining whether the duration of the shuttle capture event falls in arange between a pre-defined minimum duration and a pre-defined maximumduration.

For example, a range of 0 milliseconds to 2 milliseconds corresponds toa blockage peak at 40% current blockage; a range of 0 milliseconds to 5milliseconds corresponds to blockage peaks at 45% and 52% currentblockage; a range of 0 milliseconds to 10 milliseconds corresponds toblockage peaks at 40%, 45%, and 52% current blockage; a range of 0milliseconds to 30 milliseconds corresponds to a blockage peak at 57%current blockage; a range of 0 milliseconds to 100 millisecondscorresponds to blockage peaks at 40%, 45%, 52%, and 57% currentblockage; a range of 0 milliseconds to 500 milliseconds corresponds toblockage peaks at 40%, 45%, 52%, and 57% current blockage; and a rangeof 0 milliseconds to 1000 milliseconds corresponds to a blockage peak at80% current blockage.

Blockage levels, as identified in the PDLs, are different when just aprotein molecule is trapped by the pore from when a target molecule isattached to the protein shuttle and linked to the pore. The differencein the blockage levels can identify that a target molecule is linked.For example, when Avidin is trapped alone in ClyA, the PDL peaks are atblockage levels of 40%, 45%, 52%, and 57%, although these peaks willchange when a target molecule is attached to Avidin.

In some examples of step 624, the determining step can compriseidentifying a particular blockage event as transient if the length oftime of the particular blockage event is less than a threshold period oftime. The threshold period of time can be any period of time less than 1second.

Therefore, FIG. 6B shows how variety of abilities according to anembodiment of method 600B. For example, the different individual spectrawithin a PDL for permanent events can be identified by a length of timesince a start of the events. The PDLs for transient events, and thecomparison of PDLs for transient and permanent events, can identifyvarious trap states of a molecule or of a protein shuttle. The PDLs canalso identify an orientation of the molecule relative to the nanopore.For example, the methods of the present application identify that Avidinhas a particular orientation relative to the ClyA nanopore when Avidinis in the state AC80. The PDLs for permanent events are particularlyrelevant for analyzing a target molecule linked to a protein shuttle.When a target molecule is linked to the protein shuttle, the permanentevent PDLs will have new blockage level values and new heights of thecorresponding peaks in the individual spectra of a PDL. The heights ofthese peaks change from spectrum to spectrum within a PDL to reveal theglobal dynamics of the linked target molecule. The heights changes fromspectrum to spectrum is a result of the linked target molecule becausethe shuttle molecule is captured stably in a permanent state.

As discussed later with regards to FIGS. 14A-14G, the peak heights canbe obtained by fitting the individual spectra with sums of Gaussiandistributions.

In some examples, the method 600B can identify an expected proteinmolecule orientation based on the blockage level. This is discussedfurther with respect to FIGS. 11A-11E.

FIGS. 7A-8B show a collection of single molecule capture events in the1.66 nS ClyA nanopore. FIGS. 7A-8B show X-Y plots of the observedcurrent, I(t), during each capture event, divided by the measuredopen-nanopore current, I₀. I₀ is also referred to as the input currentfor the purposes of the present disclosure. The plotted events in FIGS.7A-7E show the time-dependent blockage for transient events ofincreasing duration. Most transient events are simple, having only asingle blockade level as in the first event in FIG. 7A. The longertransient events, though not the most common, demonstrate that sometimestransitions between levels are observed. In a first-pass step ofanalysis, each of these Transient Captures can be characterized by twonumbers, namely, the duration of the event, and the average “blockage”during the event. The “blockage” is defined to be (I₀−I(t))/I₀, averagedover each blockage event's duration.

FIGS. 8A-8B show the current traces for two Permanent Captures. Each ofthese events has an interesting time structure. As with most PermanentCapture events, they start with a preliminary, fluctuating blockage thatis followed by a deep and quiet permanent blockage. Permanent Capturesare characterized by the time duration and average blockage during thepreliminary part of the event.

In some instances, an entrance level of a protein molecule can beidentified for a particular protein molecule-nanopore combination. Forexample, Avidin has an entrance level when interacting with the ClyAnanopore. Therefore, Avidin has a particular orientation that it mustenter the nanopore in order to subsequently enter into a permanentlytrapped state.

The fact that a permanently trapped state with well-defined orientationexists is important for the shuttle platform. The state can be used totransport other molecules to the pore, where they can be stably held ina particular orientation for analysis. The PDL method can then be usedto reveal the dynamics of the analyte/target molecule thus transportedto the pore. In this situation with Avidin trapped in the pore (i.e.,the shuttle docket in the pore a stable current background is provided(which is the low-noise residual current in the stably trapped state ofAvidin). Perturbations of this current level can then be interpreted asbeing due to the analyte molecule and its dynamics which is the moleculeand dynamics of interest here. PDL plots could now be generated based onthe time-dependent current or blockage signal of an event where theinteresting current or blockage is the current or blockage differencerelative to that of the permanent level of just Avidin in the pore. Thisis discussed further with respect to FIG. 13 .

FIGS. 9A-9D show scatter plots of these parameters for each TransientCapture event (light points) reflecting this characterization. Eachlight point belongs to one protein capture. As shown in FIGS. 9A-9D, asthe Transient Capture event duration increases from a few hundredmicroseconds to a few hundred milliseconds, the average blockage slowlyincreases. This results from the existence of multiple discrete blockagelevels that are averaged over each event in the scatter plot.

Permanent Capture event are shown in the scatter plots of FIGS. 9A-9D asthe darker points. FIGS. 9A-9D suggest that the average blockage isclearly independent of duration and is close to that of the longesttransient events. This suggests that prior to capture to a deeppermanent blockage level, the protein often passes through anintermediate state of variable duration.

The circled regions in FIGS. 9A, 9C, and 9D show the noisy nature of thedata collection. These events have a blockage level of 20-30% which islower than would be expected if a protein shuttle bonded to thenanopore. Therefore, the data points in the circled region most likelyreflect molecular fluctuations in the structure of the nanopore. In someof these instances, the nanopore system can be reset so that thenanopore returns to its original configuration with a proper inputcurrent. These data points are excluded from data analysis.

FIGS. 10A-10F show exemplary PDL histogram plots, according to anembodiment of the present disclosure. The existence of discrete blockagelevels and their time-dependent populations are shown dramatically andquantitatively in the Protein Dynamics Landscape (PDL) plots in FIGS.10A-10F. These plots can be generated according to the methodologydiscussed with respect to FIG. 6A. In FIGS. 10A, 10C, and 10E, onlytransient events that last for times less than a specified Ttrans areplotted. The amount of time spent (in units of 160 microseconds) at eachblockage level for all these events is determined and a histogram of thetime spent at each level is generated. This histogram can be referred toas a blockage spectrum. A collection of such blockage spectra, for a setof increasing Ttrans values, can then be termed a Protein DynamicsLandscape (PDL). For example, Ttrans can be 1 second.

The PDLs for FIGS. 10A-10B are obtained for a fixed bias voltage of −30mV, FIGS. 10C-10D have a fixed bias voltage of −35 mV, and FIGS. 10E-10Fhave a fixed bias voltage of −40 mV. The PDL plots reveal several peaks,corresponding to discrete blockage levels for Avidin in ClyA. Note thatdeeper blockage levels last longer than shallower levels.

Permanent capture event data are provided in FIGS. 10B, 10D, and 10F. Inthese graphs, all permanent capture events are considered together andeach histogram refers to the accumulated time (in units of 160microseconds) a blockage value appears up until τ_(perm) capture eventsstart (which is when Avidin first enters the nanopore). The deepestblockage level is revealed to be maximally populated for the longesttimes.

FIG. 10C shows a single peak centered at 40% blockage (Avidin Capture)AC40 for events lasting for 2 ms or less. FIGS. 10A and 10E provideplots like this for −40 mV and −30 mV. As longer events are included(i.e., as Ttrans is increased), a second peak emerges. With carefulfitting, this second peak is centered at 52% blockage and denotes thelevel AC52. At 10 ms, the two peaks have equal intensity, and at 30 ms,the AC52 peak dominates. For longer times, a larger and separate (AC57)peak at 57% blockage dominates. FIG. 10A shows a few very deep capturesfor transient events that last longer than ½ second. Here the blockagelevel at 80% dominates (AC80). Gaussian fits to the spectra clearlyreveal a fifth peak at 45% blockage (AC45), and the fits are shownfurther in FIGS. 13B-13C.

The dynamic behavior of the transient event PDL plots can be compared tothe permanent event PDL plots of FIGS. 10B, 10D, and 10F. During thefirst 2 ms of the permanent events, the spectrum is dominated by onepeak: AC57. The AC40 peak is barely visible, and AC52 is missing. Thespectra are generally dominated by AC57 during the early times of thepermanent events, but later (>=100 ms) the AC80 peak dominates.

Table 2, below, shows the number of transient events for each blockagespectrum of FIG. 10A. Total number of permanent events at −30 mV is 131.

TABLE 2 Total Number of Transient Events at −30 mV. τ_(trans) 2 ms 5 ms10 ms 30 ms 100 ms 500 ms 1000 ms Number 566 943 1205 1552 1781 18541861 of transient events

Table 3, below, shows the number of transient events for each blockagespectrum of FIG. 10E. Total number of permanent events at −40 mV is 139.

TABLE 3 Total Number of Transient Events at −40 mV. τ_(trans) 2 ms 5 ms10 ms 30 ms 100 ms 500 ms 1000 ms Number 20 35 48 73 88 93 94 oftransient events

Therefore, FIGS. 10A-10F show how the PDL plots can reveal indicationsof the protein behavior. FIGS. 11A-11B show the expected Avidinorientation that corresponds to the AC40 states. FIG. 11C shows theexpected Avidin orientation that corresponds to the AC52 state. FIG. 11Dshows the expected Avidin orientation that corresponds to the AC57state, and FIG. 11E shows the expected Avidin orientation thatcorresponds to the AC80 state. Therefore, FIGS. 10A-10F indicate thatfor the permanent trapping events, Avidin is captured into state AC57shown in FIG. 11D, where it stays for an average of 60 ms (at −35 mV).This is followed by capture into a deeply and permanently trapped state,AC80, shown in FIG. 11E. This scenario is supported by the experimentaltime traces for the Permanent Capture events shown in FIGS. 8A-8B. Thepresent methodology can also observe two weak, additional peaks in thePDLs at other bias voltages which correspond to rarely encountered,discrete levels at 66%, at AC66, and 90% blockage, respectively.

For the 1.66 nS pore FIGS. 10A-10F show that the level populations inthe PDL's are also strongly voltage-bias dependent. This effect isdramatically indicated by plotting the ratio of the number of Permanentevents to the number of Transient events as a function of applied biasvoltage. This is shown in FIG. 12 . Increasing the bias voltage from 30mV to 50 mV results in dramatic increase in this ratio. At high biasvoltage, the energy landscape of the protein in the pore is clearlybiased towards permanently capturing the Avidin to the deepest level inthe pore from which escape eventually becomes impossible.

FIG. 12 demonstrates that the PDL method of analysis provided hereinenables the revealing of details that are very specific to particularproperties, such as orientation of the protein in the nanopore as wellas target molecules shuttled to the nanopore by the protein. Togetherwith the experimental methods provided herein, the analysis methodthereby provides a powerful technique for achieving single-moleculecharacterization with the nanopore-based platform provided herein.

For the Avidin-biotin complex, the current blockage for the deeplytrapped level is decreased by 4% relative to that for Avidin alone. Thisis shown in the plot in FIG. 13 . Since the deep blockage level forAvidin is independent of voltage in the −30 mV to −35 mV range, thedifference in deep blockage levels can be due to a slightly bigger sizefor the biotin-Avidin complex.

FIGS. 14A-14G show exemplary Gaussian fits to the PDL histogram plots,according to an embodiment of the present disclosure. The positions ofdiscrete blockage levels can be obtained from fits to blockage spectrain a PDL plot. For the PDL plot for transient events at −35 mV (FIG. 10Cabove), the levels below the 80% permanent level (AC80) can be obtainedby fitting a sum of four Gaussians to each spectrum for blockages below65%. FIGS. 14A-14G show exemplary Gaussian fits to the PDL histogramplots of FIGS. 10A-10F.

To produce the efficient convergence of the fitting routine of FIGS.14A-14G, the following procedure can be used: First the spectrum forTtrans=1000 ms is considered. One Gaussian can be fitted to the spectrumin a restricted blockage region around the dominant 57% peak, and thensubtract the resulting Gaussian from the data. The result of thissubtraction shows the 52% peak clearly. Another Gaussian can be fittedin a blockage region around that peak and again the resulting Gaussiancan be subtracted. This reveals the 45% peak which is fitted with aGaussian in a restricted blockage region. The resulting values for theposition and width of the two Gaussians centered at 57% (AC57) and 45%(AC45) are then used as fixed values for fits with four Gaussians to allspectra over the whole blockage region below 65%. The result of the fitsand the values of positions, widths, and amplitudes of each Gaussiancomponent are shown in FIGS. 14A-14G.

The results of the fitting procedure for the position (Pos), width(Sigma), and amplitude (Amp) of each peak and for each spectrum areshown in Table 3, below.

TABLE 3 Amplitude, Position, and Widths for Gaussian Distributions ofPDL Plots AC40 AC45 AC52 AC57 τ_(trans) Amp Pos Sigma Amp Amp Pos SigmaAmp 1000  817 ± 434 0.4001 ± 0.012 0.0178 ± 0.012 1792 ± 3068 ± 0.52720.0371 20422 ± 373 411 351  500  847 ± 251 0.4024 ± 0.008 0.0194 ± 0.0081531 ± 3818 ± 0.5272 0.0371 16914 ± 232 285 213  100 809 ± 78 0.4080 ±0.004 0.0240 ± 0.004 808 ± 3561 ± 0.5272 0.0371 4506 ± 74 165 69  30 644± 28 0.4069 ± 0.002 0.0247 ± 0.001 537 ± 1793 ± 0.5181 0.0333 1069 ± 2361 23  10 370 ± 12 0.4058 ± 0.001 0.0248 ± 0.001 228 ± 468 ± 0.51060.0321  185 ± 10 27 10   5 207 ± 9  0.4012 ± 0.001 0.0240 ± 0.001 136 ±172 ± 0.5106 0.0321  44 ± 7 15 8   2 69 ± 3 0.3996 ± 0.001 0.0248 ±0.001 33 ± 29 ± 0.5106 0.0321  3 ± 2 5 2

The entrance level can be first identified from a plurality of theindividual permanent event's current signatures. Then in the PDL forpermanent events, the presence of the entrance level is confirmed by theappearance of a peak at lower blockage level (i.e., 57% for Avidin) thanthe 80% permanent blockage level (also for Avidin). The presence of thislower peak in all spectra within a permanent-event PDL for Avidin, andthe absence of the other peaks found in the transient-event PDL (thepeaks at 40%, 45%, 52%) prompts the conclusion that a particularorientation of Avidin relative to the nanopore is required for permanenttrapping. (The position of the peak positions are determined by fittingwith sums of Gaussians to individual spectra within a PDL). These levelsand conclusion are for Avidin in ClyA, which are just one example ofusing the PDL analysis and visualization of the dynamics and orinteraction of proteins.

EXAMPLES

ClyA Monomer Expression and Purification

All reagents can be purchased from Fisher Scientific and/or BostonBioproducts unless otherwise stated. Phenylmethane sulfonyl fluoride(PMSF) and magnesium chloride were purchased from Sigma. The gradient4-15% gels were purchased from Bio-rad and the detergentn-Dodecyl-β-D-maltopyranoside (DDM) was purchased from EMD Millipore.

C-terminal His6 tagged ClyAwt protein was expressed in BL21 (DE3) cells.Specifically, pT7-ClyAwt-CHis6 plasmid was transformed in BL21 (DE3)chemically competent cells and grown on LB-Amp Agar plates. One colonywas inoculated in starter LB media containing 100 μg/ml ampicillinantibiotic and grown at 37° C. with shaking at 200 rpm. The starterculture was used to inoculate 250 ml LB media containing 100 μg/mlampicillin. The culture was grown at 37° C. until the OD600 was between0.5 and 0.65. The culture was then cooled on ice and induced by addingIPTG to a final concentration of 0.5 mM and then incubated for 16 hrs at15° C. with shaking. After 16 hrs, the culture was harvested at 3100×gand the pellet re-suspended in 15 ml of 50 mM Tris-HCl pH 8.0, 1 mM EDTAbuffer and frozen in −20° C. until ready to use.

The frozen pellet was subsequently thawed at room temperature and afinal concentration of 0.5 mM PMSF was added. The mixture was sonicatedon ice to lyse the cells. MgCl2 was added to the lysate at a finalconcentration of 10 mM and the mixture was then centrifuged for 20 minsat 20,000×g. The supernatant was filtered through a 0.22 μm filtermembrane and loaded onto a gravity NiNTA column equilibrated with bufferA (150 mM NaCl, 50 mM Tris-HCl pH 8). The column was then subsequentlywashed with buffer A to remove unbound proteins. Buffer A1 (150 mM NaCl,50 mM Tris-HCl, 50 mM imidazole) was used to wash the weakly boundproteins and then the ClyA protein was eluted and collected in buffer A2(150 mM NaCl, 50 mM Tris-HCl, 150 mM imidazole).

The eluted ClyA proteins were dialyzed using a 6-8 kDa cutoff membranewith constant stirring at 4° C. for two cycles in dialysis buffer (150mM NaCl, 50 mM Tris-HCl, 5 mM EDTA). The proteins were then concentratedusing a 10 kDa cutoff centricon to ˜3 ml and loaded onto a gelfiltration column equilibrated in 150 mM NaCl, 20 mM sodium phosphate pH7.0 buffer to remove aggregated proteins. The ClyA monomer was collectedand kept at 4° C. for 2 weeks or in −80° C. for long-term storage.

Preparation and Purification of ClyA Oligomers (Nanopores)

Purified ClyA monomers were next suspended at 0.6 mg/mL in a bufferedsolution containing 50 mM NaCl, 10 mM sodium phosphate pH 7.4 (with abuffer exchange column). Oligomeric ClyA was formed from monomers by theaddition of n-Dodecyl beta-D-maltoside (DDM, Calbiochem/EMD Millipore;10% w/v in water) to a final concentration of 1% and incubated 20 min atroom temperature.

ClyA nanopore purification was carried out by blue native gelelectrophoresis using a 4-16% polyacrylamide gradient gel (NativePAGE,Invitrogen/Novex Life Technologies). Typically, 10 ug of ClyA oligomerswere combined with electrophoresis loading buffer and applied to a 1.0mm×5.0 mm sample well of the gel. Major bands of oligomeric ClyA wereexcised from the gel following electrophoresis, and nanopores wererecovered from the gel slices by diffusion into an elution buffercontaining 150 mM NaCl, 0.2% DDM, 50 mM Tris-Cl pH 8.0.

Avidin Preparation

Lyophilized purified Avidin from hen egg white (Pierce/Thermo ScientificProduct# 21121) was weighed and dissolved in deionized water to 2 mg/mLconcentration. For subsequent storage at 4C, an equal volume of 2×Phosphate Buffered Saline with 20% glycerol was added to the suspensionto bring the Avidin stock solution concentration to a nominal 1 mg/mL.Prior to use in ClyA nanopore experiments, an aliquot of the Avidinstock solution was applied to a Bio-Spin 30 spin column (Bio-RadLaboratories) equilibrated with 150 mM NaCl, 15 mM Tris-Cl pH 7.5 forbuffer exchange.

Preparation of d-Biotin

An approximately 1 mM biotin solution was prepared by dissolving 0.2 mgd-Biotin (Sigma-Aldrich/Millipore Sigma) per mL of 20 mM KCl, 50 mMTris-Cl pH 7.5.

The nanopore-matched protein shuttle described above is not limited tothe Avidin-ClyA protein-nanopore example. The study of protein andtarget molecule structure, dynamics, and enzymatic activity moregenerally is enabled herein by a nanopore that has an inner lumendiameter that is substantially the same or less than the diameter of aprotein, for some protein configuration and for at least one site alongthe nanopore length. Permanent capture of the protein in the nanopore isenabled herein and can be exploited for study of the protein and targetmolecules provided in the molecular shuttle. In a correspondingmethodology, a protein shuttle and a target molecule are captured at orat least partially in a nanopore, studied, and then ejected from thenanopore. The low current noise observed for deeply-trapped Avidin andan Avidin-biotin complex indicates that this system provides a quietbackground for studying electrical signals that are induced by thetarget molecule and/or substrates interacting with the target moleculeand/or products produced by such interaction. An Avidin-biotin complexemployed in a molecular shuttle thereby enables control of targetmolecule study at a level heretofore unachievable.

While various examples of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedexamples can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described examples. Rather, the scope of the invention should bedefined in accordance with the following claims and their equivalents.

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The terminology used herein is for the purpose of describing particularexamples only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” or variants thereof, are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

What is claimed is:
 1. A method for characterizing shuttle capture in ananopore sensor, comprising: collecting a plurality of time-dependentcurrent blockage signatures for a plurality of different bias voltages;classifying the plurality of time-dependent current blockage signaturesinto permanent event signatures and transient event signatures; for eachof the plurality of bias voltages, generating a protein dynamicslandscape (PDL) for the transient event signatures, wherein the PDLcomprises a collection of blockage spectra, wherein each of thecollection of blockage spectra comprises a histogram of blockage valuesfor events of a selected maximum duration; identifying a permanent levelblockage value based on the permanent event signatures; selecting one ofthe plurality of bias voltages corresponding to a PDL for the transientevent signatures where the collection of blockage spectra reveals peaksat blockage values that are substantially equal to the permanent levelblockage value.
 2. The method of claim 1, wherein the time-dependentcurrent blockage signature comprises a measurement of nanopore currentas a function of time during a shuttle capture event.
 3. The method ofclaim 1, wherein the classifying further comprises identifying as thetransient event signatures any one of the plurality of time-dependentcurrent blockage signatures showing a return to an initial currentlevel.
 4. The method of claim 3, wherein the classifying furthercomprises identifying as the permanent event signatures any one of theplurality of time-dependent current blockage signatures failing to showa return to an initial current level.
 5. The method of claim 4, furthercomprising: for each of the plurality of bias voltages, calculating aratio of a number of permanent shuttle capture events to a number oftransient shuttle capture events; and based on the calculated ratios,determining a threshold bias voltage to achieve a preferred ratio. 6.The method of claim 1, wherein the blockage spectra further comprises anaverage of nanopore current blockage data, sampled over a fixed timeinterval to yield histogram data points.
 7. The method of claim 6,wherein the fixed time interval is at least as large as an interval forsampling the nanopore current blockage data.
 8. The method of claim 6,wherein the nanopore current blockage data comprises a ratio of a changein the current from an input current to the input current.
 9. The methodof claim 1, wherein the identifying comprises: generating an additionalPDL for the permanent event signatures for at least one of the pluralityof bias voltages; and selecting an entrance level blockage value tocorrespond to a blockage level lower than the permanent blockage level.10. The method of claim 9, wherein the method further comprises: fittingeach spectrum of the collection of blockage spectra within a PDL with asum of Gaussian distribution; determining the entrance level blockagevalue from a peak in the collection of blockage spectra within the PDLfor permanent events.
 11. The method of claim 1, wherein the selectedmaximum duration of the particular blockage spectra is at least one of:2 milliseconds, 5 milliseconds, 10 milliseconds, 30 milliseconds, 100milliseconds, 500 milliseconds, and 1000 milliseconds.